Anti-human alpha9 integrin antibody and use thereof

ABSTRACT

The present invention relates to an anti-human α9 integrin antibody. More particularly, the present invention relates to: a monoclonal antibody, a chimeric antibody, a humanized antibody and a human antibody that specifically recognize human α9 integrin; a hybridoma cell that produces the monoclonal antibody; a method for producing the monoclonal antibody; a method for producing the hybridoma cell; a therapeutic agent comprising the anti-human α9 integrin antibody; a diagnostic agent comprising the human α9 integrin antibody; and a method for screening for a compound that inhibits the activity of human α9 integrin.

TECHNICAL FIELD

The present invention relates to an anti-human α9 integrin antibody anduse thereof. More particularly, the present invention relates to: amonoclonal antibody, a chimeric antibody, a humanized antibody and ahuman antibody that specifically recognize human α9 integrin; ahybridoma cell that produces the monoclonal antibody; a method forproducing the monoclonal antibody; a method for producing the hybridomacell; a therapeutic agent comprising the anti-human α9 integrinantibody; a diagnostic agent comprising the anti-human α9 integrinantibody; and a method for screening for a compound that inhibits theactivity of human α9 integrin.

BACKGROUND ART

Adhesion between a cell and the extracellular matrix is mediated by atransmembrane cell adhesion protein as typified by integrins. Integrinsare heterodimers that have 1:1 of α- and β-chains. So far, 18 types ofα-chains and 8 types of β-chains have been found where at least 24 typesof combinations thereof have been identified and confirmed. Eachintegrin is known to recognize a specific extracellular matrix (ligand).Moreover, the transmembrane cell adhesion protein containing an integrinnot only plays a role of adhering and anchoring a cell and theextracellular matrix but it has also been found to convert informationfrom the extracellular matrix into intracellular signals for regulatingcell proliferation, motility, cell death, differentiation and the like.

Integrins are classified, according to their specificities and functionswith respect to ligands, into subfamilies, namely, collagen receptors,laminin receptors, RGD receptors that recognize Arg-Gly-Asp (RGD)sequence contained in fibronectin, vitronectin and the like, andleukocyte-specific receptors that exist only in leukocytes. Alpha-4integrins and α9 integrins do not belong to any of these subfamilies andare called α4 integrin subfamily.

Ligands that are known to bind to α4 and α9 integrins includeosteopontin (hereinafter, referred to as OPN), EDA domain offibronectin, propeptide-von Willebrand factor (pp-vWF), tissuetransglutaminase (tTG), coagulation factor XIII and Vascular CellAdhesion Molecule-1 (VCAM-1). Furthermore, ligands that are known to berecognized specifically by α4 integrin include CS-1 domain offibronectin, MadCAM-1 (α4β7) and the like. Meanwhile, ligands that areknown to be recognized specifically by α9 integrin include tenascin C,plasmin and the like.

OPN, one type of extracellular matrices (ECM), is a secreted acidphosphorylated acid glycoprotein with a molecular weight of about 41kDa, which is a molecule generally acknowledged to be expressed inbreast milk, urine, renal tubules, osteoclasts, osteoblasts, macrophage,activated T cell, tumor tissue and the like. OPN has cell adhesionsequence GRGDS in the middle, and SVVYGLR or SLAYGLR sequence for humanor murine OPN, respectively, which is immediately followed by a thrombincleavage site. OPN adheres to integrin of RGD receptor via the GRGDSsequence and to α4 (α4β1) and α9 (α9β1) integrins via the SVVYGLRsequence or the SLAYGLR sequence.

While α4β1 binds to OPN that is not cleaved with thrombin (non-cleavedOPN) as well as an N-terminal fragment cleaved with thrombin (cleavedOPN), α9β1 differs in that it binds only to cleaved OPN.

The amino acid sequences of α4,α9 and β1 integrin subunits are known andare registered with GenBank. The amino acid sequences are known to behighly similar among the species.

WO02/081522 discloses a therapeutic effect for rheumatoid arthritis andhepatitis by utilizing an OPN-deficient mouse and a neutralizingantibody against OPN to suppress the OPN functions. This publicationalso discloses that the SVVYGLR sequence, i.e., a sequence thatrecognizes α4 and α9 integrins, plays an important role upon onset of aninflammatory disease and that a receptor for OPN involved ininflammatory diseases is expressed in the immunocompetent cell or thelike.

-   [Patent Document 1] WO02/081522

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Currently, various therapeutic drugs for cancers, inflammatory diseases,infectious diseases, autoimmune diseases and bone diseases are known butdevelopment of a prophylactic drug and/or a therapeutic drug withimproved therapeutic effects for cancers, inflammatory diseases,infectious diseases, autoimmune diseases and bone diseases are desired.

Until today, the present inventors have conducted various studiesfocusing on integrins, in particular α9 integrin. As a result, theyfound that an antibody that specifically inhibits α9 integrin has acancer suppressing effect as well as an anti-inflammatory effect andproduced five types of hybridoma cells that produces the monoclonalantibody (1K11, 21C5, 24I11, 25B6 and 28S1) (each deposited with theInternational Patent Organism Depositary, the National Institute ofAdvanced Industrial Science and Technology (Central 6, Higashi 1-1-1,Tsukuba-shi, Ibaraki-ken, 305-8566) as FERM BP-10510, FERM BP-10511,FERM BP-10512, FERM BP-10513 and FERM BP-10832, respectively (the firstfour cells deposited on Feb. 15, 2006 and the last one on May 29,2007)).

Under such circumstances, there has been a need for a monoclonalantibody or an alternative monoclonal antibody with higher drugefficacy.

Means for Solving the Problem

In order to develop a monoclonal antibody or an alternative monoclonalantibody that has superior drug efficacy over the above-mentioned fivemonoclonal antibodies, the present inventors have gone through keenstudies and succeeded in producing a hybridoma cell that produces such anovel monoclonal antibody, thereby accomplishing the present invention.

Thus, the present invention provides an anti-human α9 integrin antibody,a monoclonal antibody thereof, a cell that produces the antibody, atherapeutic agent comprising the antibody, and a method for screeningfor a compound that inhibits α9 integrin activity.

(1) An anti-human α9 integrin antibody comprising an amino acid sequencerepresented by any one of SEQ ID NOS:1-12.(2) An anti-human α9 integrin antibody comprising an amino acid sequencerepresented by SEQ ID NO:1, 3, 5, 7, 9 or 11.(3) An anti-human α9 integrin antibody comprising amino acid sequencesrepresented by SEQ ID NOS:1, 3, 5, 7, 9 and 11.(4) An anti-human α9 integrin antibody comprising an amino acid sequencerepresented by SEQ ID NO:2, 4, 6, 8, 10 or 12.(5) An anti-human α9 integrin antibody comprising amino acid sequencesrepresented by SEQ ID NOS:2, 4, 6, 8, 10 and 12.(6) An anti-human α9 integrin antibody comprising the amino acidsequence represented by any one of SEQ ID NOS:1-6 as an amino acidsequence of the complementary determining region of the heavy chain(CDRH) and the amino acid sequence represented by any one of SEQ IDNOS:7-12 as an amino acid sequence of the complementary determiningregion of the light chain (CDRL).(7) The anti-human α9 integrin antibody according to any one of (1)-(6)above, wherein the binding between the human α9 integrin and the ligandof α9 integrin is inhibited.(8) The anti-human α9 integrin antibody according to any one of (1)-(7)above, wherein it is a monoclonal antibody.(9) The anti-human α9 integrin antibody according to any one of (1)-(8)above, wherein it is a chimeric antibody.(10) The anti-human α9 integrin antibody according to any one of (1)-(8)above, wherein it is a humanized antibody.(11) The anti-human α9 integrin antibody according to any one of (1)-(8)above, wherein it is a human antibody.(12) An anti-human α9 integrin antibody produced by a hybridoma cellassigned Accession No. FERM BP-10830 or FERM BP-10831.(13) A therapeutic agent for a cancer, an inflammatory disease, aninfectious disease, an autoimmune disease or a bone disease, comprisingthe anti-human α9 integrin antibody according to any one of (1)-(12)above as an active component.(14) A therapeutic agent for a cancer, an inflammatory disease, aninfectious disease, an autoimmune disease or a bone disease, comprisingthe anti-human α9 integrin antibody according to any one of (1)-(12)above as well as an anti-human α4 integrin antibody as activecomponents.(15) A diagnostic agent for a cancer, an inflammatory disease, aninfectious disease, an autoimmune disease or a bone disease, comprisingthe anti-human α9 integrin antibody according to any one of (1)-(12)above as an active component.(16) A cell that produces the anti-human α9 integrin antibody accordingto any one of (1)-(12) above.(17) A hybridoma cell assigned Accession No. FERM BP-10830 or FERMBP-10831.(18) A method for screening for a compound that inhibits an activity ofα9 integrin, comprising using a peptide comprising the amino acidsequence of α9 integrin.

Effect of the Invention

The present invention provides a novel anti-α-integrin antibody. Theanti-α9-integrin antibody of the present invention shows an excellentsuppression effect against a function of α9 integrin and exerts atherapeutic effect against cancers (for example, cancer cellproliferation or metastasis), inflammatory diseases (for example,rheumatoid arthritis, osteoarthritis, hepatitis, bronchial asthma,fibrosis, diabetes, arterial sclerosis, multiple sclerosis, inflammatorybowel diseases (ulcerative colitis, Crohn's disease, etc.)), infectiousdiseases (for example, hepatitis), autoimmune diseases (for example,systemic lupus erythematosus, polymyositis, autoimmune thyroid disease,tubulointerstitial nephritis, myasthenia gravis), bone diseases (forexample, osteoporosis) and the like. Moreover, a pharmaceuticalcomposition comprising the anti-α9-integrin antibody of the presentinvention as well as the anti-α4-integrin antibody exerts an improvedtherapeutic effect against cancers, inflammatory diseases and the like.The antibody of the present invention may also be utilized as adiagnostic drug since it is capable of pathologically detectingexpression of α9 integrin in cells and tissues.

MODES FOR CARRYING OUT THE INVENTION Background of the Invention

Tysabri (registered trademark) (natalizumab), an antibody against α4integrin, from Biogen Idec Inc. (Massachusetts, USA) and ElanCorporation (Ireland), was approved as a drug for multiplesclerosistherapeutic by the Food and Drug Administration (FDA) inNovember 2004. Furthermore, Tysabri (registered trademark) is undergoingclinical development directing at diseases such as Crohn's disease andrheumatoid arthritis. An anti-human α4β1 integrin monoclonal antibody,called P4C2, is used in the laboratory.

Although a monoclonal antibody named Y9A2 that shows specificity tohuman and guinea pig α9 integrins (A. Wang et al, (1996) Am. J. Respir.,Cell Mol. Biol. 15, 664-672) has been subjected to experiments as anantibody against α9 integrin, its clinical use has not yet beenrealized.

According to the present invention, a novel antibody against human α9integrin, which is expected to have superior drug efficacy, was obtainedby carefully pursuing the following procedures.

(1) Production of Human α9 Integrin Overexpressing Strain

In order to produce an antibody against α9 integrin, gene transfectionof mouse fibroblast NIH-3T3 cell was performed to establish a cell linethat overexpresses human α9 integrin. This cell line is used as anantigen for immunizing mice.

(2) Screening for Hybridoma

In order to efficiently obtain clones that react exclusively to human α9integrin from various hybridoma resulting from cell fusion, human α4integrin belonging to the same integrin family is expressed in CHO-K1cells. The resulting cells are used to select clones that do not showcross reactivity with other integrins and that do not react with thecell surface antigen of the parent cell (CHO-K1), thereby efficientlyobtaining an inhibition antibody that reacts specifically to human α9integrin.

Anti-α9-Integrin Antibody of the Present Invention

The present invention provides a monoclonal antibody against human α9integrin. According to the present invention, the term “antibody” refersto the whole antibody molecule or a fragment thereof (for example, Fab,Fab′ or F(ab′)₂ fragment) that specifically binds to α9 integrin or thepartial peptide thereof as an antigen, which may be either a polyclonalantibody or a monoclonal antibody. According to the present invention,the term preferably refers to a monoclonal antibody. The term “antibody”according to the present invention also encompasses a chimeric antibody,a humanized antibody and a human antibody.

According to the present invention, when an antibody “specificallybinds” to a certain protein or a fragment thereof, it means that theantibody binds to a particular amino acid sequence of the certainprotein or the fragment thereof with substantially higher affinity overother amino acid sequences. Herein, the phrase “substantially highaffinity” refers to a level of affinity that is sufficient todistinguish and detect a particular amino acid sequence from other aminoacid sequences with a desired measurement device or method. Typically,the phrase refers to binding affinity with an association constant(K_(a)) of at least 10⁷M⁻¹, preferably at least 10⁸M⁻¹, more preferably10⁹M⁻¹, still more preferably 10¹⁰M⁻¹, 10¹¹M⁻¹, 10¹²M⁻¹ or higher, forexample, up to 10¹³M⁻¹ or higher.

A “monoclonal antibody” according to the present invention refers to anantibody that is highly specific to recognize a single antigen.

According to the present invention, an “antibody fragment” refers to apart of a whole antibody, namely, an antigen-binding region or avariable region. Examples of antibody fragments include Fab, Fab′,F(ab′)₂ and Fv fragments. These antibody fragments may be producedthrough a generally known technique such as papain digestion or pepsindigestion of the antibody.

The above-mentioned “chimeric antibody” refers to a chimeric human-mouseantibody obtained by genetically engineering the constant region of theanti-human α9 integrin antibody of the present invention to have thesame constant region as that of the human antibody (see European PatentPublication No. EP0125023). A “humanized antibody” refers to an antibodyobtained by genetically engineering the primary structure of theanti-human α9 integrin antibody of the present invention to have thecorresponding primary structure of a human antibody except for thecomplementarity determining regions (CDRs) of the H- and L-chains. A“human antibody” refers to a monoclonal antibody produced by using atransgenic animal that has been transferred with a human gene involvedin human antibody production (see European Patent Publication No.EP0546073).

More specifically, the present invention provides an anti-human α9integrin antibody that differs from the conventionally-producedanti-human α9 integrin antibodies. An antibody according to thepreferred embodiment of the present invention comprises an amino acidsequence represented by SEQ ID NO:1, 3, 5, 7, 9 or 11. More preferableantibody is an anti-human α9 integrin antibody comprising two or more,three or more, four or more, five or more or six amino acid sequencesselected from the group consisting of the amino acid sequencesrepresented by SEQ ID NOS:1, 3, 5, 7, 9 and 11.

Furthermore, an antibody according to another embodiment of the presentinvention comprises an amino acid sequence represented by SEQ ID NO:2,4, 6, 8, 10 or 12. More preferable antibody is an anti-human α9 integrinantibody comprising two or more, three or more, four or more, five ormore or six amino acid sequences selected from the group consisting ofthe amino acid sequences represented by SEQ ID NOS:2, 4, 6, 8, and 12.

Particularly preferable antibody of the present invention is ananti-human α9 integrin antibody produced by a hybridoma cell assignedAccession No. FERM BP-10830 or FERM BP-10831.

Hereinafter, production of an anti-α9-integrin monoclonal antibody willbe described, although the production of the antibody should not belimited thereto.

Alpha-9 Integrin (Antigen)

Alpha-9 integrin as an antigen used with the present invention may be(1) any cell derived from human or other mammal that expresses α9integrin or a protein derived from any tissue containing this cell, (2)gene DNA coding for α9 integrin, preferably a recombinant protein inwhich cDNA is introduced and expressed in a cell line such as abacterium, an yeast or an animal cell, or (3) a synthetic protein.

Furthermore, α9 integrin of the present invention also comprises apolypeptide having an amino acid sequence of α9 integrin from any typeof mammal, particularly preferably a polypeptide having substantiallythe same amino acid sequence as the amino acid sequence (SEQ ID NO:13)of human α9 integrin.

Here, the phrase “a polypeptide having substantially the same amino acidsequence” refers to a mutant polypeptide having an amino acid sequencewith substantially the same biological nature as that of the amino acidsequence of natural α9 integrin, particularly preferably human-derivedα9 integrin, where several amino acids, preferably 1-10 amino acids andparticularly preferably one to several (for example, 1-5, 1-4, 1-3 or1-2) amino acids are substituted, deleted and/or modified in said aminoacid sequence, as well as a mutant polypeptide having an amino acidsequence having several amino acids, preferably 1-10 amino acids,particularly preferably one to several (for example, 1-5, 1-4, 1-3 or1-2) amino acids added to the amino acid sequence of natural α9integrin, particularly preferably human-derived α9 integrin. Thepolypeptide may also be a mutant polypeptide further having some of suchsubstitution, deletion, modification and addition.

Alpha-9 integrin, particularly human-derived α9 integrin, of the presentinvention may be produced by appropriately employing a gene recombinanttechnique as well as a method known in the art such as chemicalsynthesis, cell culturing, or a modified method thereof.

Examples of methods for producing a mutant polypeptide includesite-directed mutagenesis using a synthetic oligonucleotide (gappedduplex approach), introduction of random point mutation using nitrousacid or sulfurous acid treatment, production of a deficient mutantusing, for example, Ba131 enzyme or the like, cassette mutagenesis,linker scanning technique, misincorporation technique, mismatch primertechnique and synthesis of DNA segment.

In addition, α9 integrin of the present invention also comprises a“part” of said α9 integrin. Herein, the term “part” refers to a portionthat includes a region necessary for binding to an α9 integrin ligandsuch as OPN, tenascin C or VCAM-1. The “part” of said a9 integrin may beproduced by a later-described gene recombinant technique or chemicalsynthesis known in the art or a modified version thereof. Alternatively,it may be produced by appropriately cleaving α9 integrin, particularlypreferably human-derived α9 integrin, that has been isolated throughcell culturing with protease or the like.

An antigen may also be a cell itself that overexpresses α9 integrin onthe cell membrane by recombinant technique, or a membrane fraction orthe like thereof.

Alpha-9 integrin of the present invention also comprises a polypeptidehaving substantially the same amino acid sequence as the amino acidsequence (SEQ ID NO:13) of human α9 integrin. According to the presentinvention, a cell itself that overexpresses human α9 integrin on thecell membrane by a recombinant technique is particularly preferablyused. Hence, a later-described cell that overexpresses human α9 integrinon the cell membrane or a cell membrane fraction thereof may be preparedto be used by itself as an antigen by cloning a gene (for example, cDNA)coding for human α9 integrin according to a known genetic engineeringtechnique.

Preparation of Antibody-Producing Cell

The antigen is administered alone or together with a carrier or adiluent to an animal to be immunized at a site that allows production ofthe antibody upon administration. In order to enhance the antibodyproducing capability upon administration, a complete Freund's adjuvantor an incomplete Freund's adjuvant may be administered. Theadministration takes place generally once in every 1-6 weeks for a totalof about 2-10 times. Examples of the warm-blooded animal used include amouse, monkey, rabbit, dog, guinea pig, rat, hamster, sheep, goat andchicken although a mouse is preferably used in the present invention.

When the subject of the treatment is human and the animal for producingthe α9 integrin-inhibiting antibody is a mouse, a chimeric antibody fromhuman and mouse or a humanized antibody is preferably used. Preferably,a transgenic animal such as a mouse that has been introduced with ahuman gene involved in antibody production is used to produce ahumanized monoclonal antibody to be employed.

Cell Fusion of Antibody-Producing Cell and Myeloma Cell

As a myeloma cell, a cell derived from a mouse, rat, human or the likeis used. Examples include mouse myeloma P3U1, P3X63-Ag8, P3X63-Ag8-U1,P3NS1-Ag4, SP2/0-Ag14 and P3X63-Ag8-653. Preferably, theantibody-producing cell and the myeloma cell are derived from animals ofthe same species, particularly animals of the same strain. The myelomacell may be cryopreserved, or maintained by passage in a general mediumsupplemented with a fetal horse, rabbit or bovine serum. For cellfusion, cells in logarithmic growth phase are preferably used. Accordingto the present invention, P3X63-Ag8-653 is preferably used.

Examples of a method for fusing the antibody-producing cell and themyeloma cell to form a hybridoma include a method using polyethyleneglycol (PEG), a method using Sendai virus, and a method using anelectric fusion device. For example, in the case of PEG method, amixture ratio of 1-10:1, preferably 5-10:1 of spleen cells and myelomacells are suspended in an appropriate medium or buffer containing about30-60% of PEG (average molecular weight: 1000-6000) to allow reaction ata temperature of about 25-37° C. under conditions of pH6-8 for about 30seconds to about 3 minutes. At the end of the reaction, the PEG solutionis removed and the resultant is resuspended in a medium and seeded in acell well plate to continue the culturing.

Sorting of Hybridoma Cells

A hybridoma cell that produces the monoclonal antibody can be sortedaccording to a known method or a method corresponding thereto. Ingeneral, sorting can be performed in a medium for animal cellssupplemented with HAT (hypoxanthine, aminopterin, thymidine). The mediumused for sorting and breeding may be any medium as long as it allowsgrowth of the hybridoma cell. For example, a RPMI 1640 medium containing1-20%, preferably 10-20% fetal bovine serum, a GIT medium (Wako PureChemical Industries, Ltd.) containing 1-10% fetal bovine serum, or aserum-free medium for culturing hybridoma (SFM-101, NissuiPharmaceutical Co., Ltd.) can be used. The temperature for culturing isgenerally 20-40° C., and preferably about 37° C. The time for culturingis generally 5 days to 3 weeks, and preferably 1-2 weeks. In general,culturing may be carried out under 5% CO₂.

Production of a monoclonal antibody of the present invention may beconfirmed and screened by employing a cellular ELISA technique describedin New Experimental Methods of Clinical Immunology (part 3), KagakuHyoron-sha, 1997. When the cell used for immunization is used forscreening, the background or the false positives may be expected toincrease. In this case, a clone that reacts with human α9 integrinoverexpressed in a cell other than the cell used for immunization, butthat does not react with a cell that overexpresses human α4 integrin,may be used as an anti-human α9 integrin antibody. Limiting dilutionmethod can be repeated once to five times, preferably twice to fourtimes, on such a clone to prepare a monoclonal antibody.

Separation and Purification of Antibody

The resulting antibody may be purified to homogeneity. Separation andpurification of the antibody may be performed by employing a separationand purification technique that is generally used for proteins. Forexample, but without limitation, a chromatography column such asaffinity chromatography, a filter, ultrafiltration, salting-out,dialysis, SDS polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis or the like may appropriately be selected or combined toseparate and purify the antibody (Antibodies: A Laboratory Manual. EdHarlow and David Lane, Cold Spring Harbor Laboratory, 1988). Examples ofa column used for affinity chromatography include protein A column andprotein G column. For example, a column using a protein A column may beHyper D, POROS and Sepharose F. F. (Amersham Biosciences).

Labeling of Antibody

The resulting antibody can be labeled according to various labeling (forexample, biotin label, FITC label, APC label) by a known method or withcommercially available kit. Preferably, a biotin label using Biotinlabeling kit (Dojindo Laboratories) is used in the present invention.

The thus-obtained monoclonal antibody is purified, if necessary, andthen formulated according to a common technique to be used as aprophylactic and/or therapeutic agent for cancers, inflammatorydiseases, infectious diseases, autoimmune diseases and bone diseases. Aformulation of these prophylactic and/or therapeutic agents may be aparenteral formulation such as an injectable agent or drip, or may bedevised for use as an oral formulation. For formulation, a carrier, adiluent, an additive or the like suitable for the formulation may beused within a medically and pharmaceutically acceptable range.

Pharmacological Effect of Antibody

Integrin not only plays a role of attaching and anchoring a cell and theextracellular matrix (ECM), but also been found to take a role inconverting information from the extracellular matrix into intracellularsignals for regulating cell proliferation, motility, cell death,differentiation and the like. Since the resulting monoclonal antibodycan interrupt the intracellular signaling of information from ECM byinhibiting the binding between ECM and α9 integrin, it can treatECM-related diseases. Examples of ECM and α9 ligands that are known tobind to α9 integrin include OPN, fibronectin, propeptide-von Willebrandfactor (pp-vWF), tissue transglutaminase (tTG), coagulation factor XIII,Vascular Cell Adhesion Molecule-1 (VCAM-1), tenascin C and plasmin.Cells or cancer cells expressing these ECM and α9 integrins are used toobserve binding inhibition in vitro in the presence of the resultingmonoclonal antibody, thereby determining a disease targeted by themonoclonal antibody of the present invention.

Pharmaceutical Agent Comprising Antibody

A formulation comprising an antibody (in particular, a monoclonalantibody) of the present invention as an active component may be used asa therapeutic agent or a prophylactic agent for cancers (for example,proliferation or metastasis of cancer cells), inflammatory diseases (forexample, rheumatoid arthritis, osteoarthritis, hepatitis, bronchialasthma, fibrosis, diabetes, arterial sclerosis, multiple sclerosis orinflammatory bowel diseases (ulcerative colitis or Crohn's disease)),infectious diseases (for example, hepatitis), autoimmune diseases (forexample, systemic lupus erythematosus, polymyositis, autoimmune thyroiddisease, tubulointerstitial nephritis or myasthenia gravis), bonediseases (for example, osteoporosis) and the like.

The dosage differs depending on the subject of administration, thetarget disease, conditions, the administration route and the like. Forexample, in the case where it is used for preventing and/or treating acancer patient, a single dose of generally about 0.01-20 mg/kg weight,preferably about 0.1-10 mg/kg weight and more preferably about 0.1-5mg/kg weight of the antibody of the present invention is convenientlyadministered by intravenous injection for about 1-10 times a month,preferably about 1-5 times a month. The dosage for other parenteral ororal administration may be determined in accordance with theabove-described dosage. If the condition is particularly severe, thedosage or the number of administration may be increased according to thecondition.

The antibody of the present invention may be administered per se or asan appropriate pharmaceutical composition. A pharmaceutical compositionused for the above-described administration comprises the antibody or asalt thereof as well as a pharmacologically-acceptable carrier, diluentor excipient. Such a composition is provided in a formulationappropriate for parenteral or oral administration.

Specifically, examples of compositions for parenteral administrationinclude an injectable agent, nasal drops and a suppository, where theinjectable agent includes formulations such as anintravenously-injectable agent, a subcutaneously-injectable agent,intradermally-injectable agent, an intramuscularly injectable agent andan injectable drip. Such an injectable agent is prepared according to aknown method, for example, by dissolving, suspending or emulsifying theantibody in a sterile aqueous or oily solution that is generally usablefor an injectable agent. For example, physiological saline, glucose,sucrose, mannitol or an isotonic solution containing other adjuvants maybe used as an aqueous solution for an injectable agent possibly inconjunction with an appropriate solubilizing aid such as an alcohol(e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethyleneglycol), a non-ionic surfactant [e.g., Polysorbate 80, Polysorbate20,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] orthe like. For example, sesame oil, soybean oil or the like may be usedas an oily solution possibly in conjunction with a solubilizing aid suchas benzyl benzoate, benzyl alcohol or the like. The prepared injectablesolution is usually packaged in a suitable ampule, vial or syringe. Asuppository used for rectal administration may be prepared by mixing theabove-described antibody into a usual nasal drop base or suppositorybase. Moreover, a suitable excipient may be added to the above-describedantibody to prepare a lyophilized formulation, which is dissolved ininjectable water, physiological saline or the like upon use as aninjectable solution. In general, since an oral administration of aprotein such as an antibody is believed to be difficult as it is brokendown by a digestive organ, oral administration may be possible bydevising the antibody fragment or the modified antibody fragment and theformulation thereof.

The above-mentioned parenteral pharmaceutical composition is preferablyprepared into a formulation in a dose unit adapted to the dosage of theactive element. Examples of such a dose-unit formulation include aninjectable agent (an ampule, a vial, a prefilled syringe), nasal dropsand suppository, where each dose unit of the formulation preferablycontains the above-described antibody for generally 5-500 mg, inparticular 5-100 mg for an injectable agent, and 10-250 mg for otherformulations.

Each of the above-mentioned composition may contain other active elementas long as it does not cause any unfavorable interaction uponcompounding with the above-described antibody. For example, apharmaceutical formulation of the present invention may contain ananti-human α4 integrin antibody in addition to the above-describedantibody. In this case, the mixture ratio is not particularly limitedbut, for example, the ratio of the anti-human α9 integrin antibody tothe anti-human α4 integrin antibody may be adjusted within the range of1:99-99:1.

Diagnostic Agent Comprising Monoclonal Antibody of the Present Invention

A pharmaceutical composition comprising a monoclonal antibody of thepresent invention may be used as a diagnostic agent for an inflammatorydisease such as rheumatoid arthritis or hepatitis, bronchial asthma,fibrosis, diabetes, cancer metastasis, arterial sclerosis, multiplesclerosis or granuloma, or as a diagnostic agent for suppression ofchronic rejection after organ transplantation, or for an autoimmunedisease such as a systemic autoimmune disease, erythematosus, uveitis,Behcet's disease, polymyositis, proliferative glomerulonephritis orsarcoidosis. Since the monoclonal antibody of the present invention canspecifically recognize α9 integrin, it may be used for quantitation ofa9 integrin in a test solution, and in particular for quantitationemploying sandwich immunoassay, a competition technique, an immunometrictechnique or the like. No particular condition, procedure or the like isrequired for adapting each of these immunological determinationtechniques to a measurement technique of the present invention. Themeasurement system may be configured by those skilled in the art byapplying general technical consideration to usual conditions andprocedures for each method. For details of these general technicalprocedures, reference may be made to articles, documents and the like.

Accordingly, an antibody of the present invention can be used toquantitate α9 integrin in a highly sensitive manner. Furthermore, anantibody of the present invention can be used to quantitate α9 integrinin vivo to diagnose various α9 integrin-related diseases. For example,when an increase or a decrease is detected in the α9 integrin level,diagnosis is that there is a high possibility of suffering or likelihoodof suffering an α9 integrin-related disease such as an inflammatorydisease. In addition, a monoclonal antibody of the present invention canbe used for specifically detecting α9 integrin existing in the subjectsuch as a body fluid or a tissue. It can also be used for preparing anantibody column used for purifying α9 integrin, for detecting α9integrin contained in each fraction upon purification, or for analyzingthe behavior of α9 integrin in a test cell or the like.

Method of Screening for Compound that Inhibits Activity of Human α9Integrin

An epitope on a human α9 integrin that is recognized by an antibody ofthe present invention may be utilized to screen for a compound that caninhibit the activity of human α9 integrin. Specifically, the presentinvention provides a method for screening for a low-molecular compoundthat inhibits the activity of human α9 integrin, the method comprisingusing a peptide comprising an amino acid sequence of human α9 integrin(hereinafter, referred to as “peptide A”).

According to the screening method of the present invention, for example,comparison is made between (i) a case where peptide A and a ligand ofhuman α9 integrin (for example, tenascin C, plasmin, etc.) are made tocontact and (ii) a case where peptide A, the ligand and a test compoundare made to contact. The cases (i) and (ii) are compared, for example,by determining binding between the ligand and peptide A. In order toreadily compare the bindings, a ligand that has been labeled accordingto a known technique is preferably used. A candidate compound resultingfrom such a method is tested to confirm whether or not it inhibits theactivity of human α9 integrin, thereby obtaining a compound thatinhibits the activity of human α9 integrin.

The test substance used may be a polypeptide, a protein, a non-peptidecompound derived from a living organism, a synthetic compound, amicrobiological culture, a cell extract, a plant extract, an animaltissue extract or the like, which may be either a novel compound or aknown compound.

As is the case with the antibody of the present invention, the selectedcompound may be used as a prophylactic and/or therapeutic agent forcancers, inflammatory diseases, infectious diseases, autoimmunediseases, bone diseases and the like.

Hereinafter, the present invention will be described in more detail bymeans of examples below, which do not limit the scope of the presentinvention.

EXAMPLES Example 1 Production of Antibody Against Human α9 Integrin

An antibody against human α9 integrin was produced by immunizing threeBALB/c mice as follows. First, 3×10⁶ cells/animal ofhuman-α9-integrin-expressing cells (human α9/NIH-3T3 cells) wereintraperitoneally administered to the mice, and further 3×10⁶cells/animal of human α9/NIH-3T3 cells were intraperitoneallyadministered following one and two weeks. A week later, 2×10⁶cells/animal of human α9/NIH-3T3 cells were intravenously administered.Clones that reacted with human α9/CHO-K1 cell and human melanoma cellline (G361 cells) endogenously expressing human α9 integrin but that didnot react with CHO-K1 cell expressing human α4 integrin were determinedas anti-α9-integrin antibodies. As a result, two clones of hybridomacells (K33N, M35A) that produced anti-human α9 integrin antibody wereestablished.

The thus-obtained hybridoma cells K33N and M35A were each deposited withthe International Patent Organism Depositary, the National Institute ofAdvanced Industrial Science and Technology (Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki, 305-8566) on May 29, 2007, and assigned Accession No.FERM BP-10830 and FERM BP-10831, respectively.

Example 2 Analysis of Complementarity Determining Region (CDR) ofAnti-Human α9 Integrin Antibody

mRNAs were extracted from hybridomas that produce the human α9 integrinantibodies (K33N, M35A), and subjected to reverse transcription toprepare cDNAs. These cDNAs were used as templates together with ScFvcloning primers (Light Primer Mix, Heavy Primer Mix; AmershamBioscience) for PCR to elongate and amplify the variable region of eachof the heavy and light chains of the antibodies. Subsequently, the PCRproducts were integrated into pCRII TOPO vectors by a common technique.The resultants were sequenced to determine the amino acid sequences. Theabove-described procedure was repeated for three times for eachantibody.

As a result, the amino acid sequences of the variable regions and theCDR regions of heavy and light chains of K33N and M35A were determinedas shown in FIGS. 1A and 1B. Specifically, the amino acid sequences ofthe CDR regions were as follows.

(Heavy chain) [CDRH1] K33N: SYYMN  (SEQ ID NO: 1) M35A: SYWIH (SEQ ID NO: 2) [CDRH2] K33N: WIFPGSGNTKYNEKFKGK (SEQ ID NO: 3)M35A: EINPSSGRTNFIENFETK (SEQ ID NO: 4) [CDRH3] K33N: SWVSYERGYYFDY(SEQ ID NO: 5) M35A: LAYGNYSWFAY (SEQ ID NO: 6) (Light chain) [CDRL1]K33N: RASENIYYSLA (SEQ ID NO: 7) M35A: RASETVDSYGNTFMH (SEQ ID NO: 8)[CDRL2] K33N: NANSLED (SEQ ID NO: 9) M35A: LASNLES (SEQ ID NO: l0)[CDRL3] K33N: KQAYDVPYT (SEQ ID NO: 11) M35A: QQNNEDPYT (SEQ ID NO: 12)

FIGS. 1A and 1B also show sequences (JN Bio and Takara) obtained bydifferent analysis methods (using different sequence analysis softwares)from the above-described sequence analysis method (GTS). The details ofeach method are described below.

Sequence Analysis Method by JN Biosciences (K33N)

The hybridoma cell (K33N) was cultured in TIL Media I medium(Immuno-Biological Laboratories) containing 10% fetal bovine serum (FBS;HyClone) under 7.5% CO₂ at 37° C. for amplification. Total RNA wasextracted from about 3×10⁶ hybridoma cells using TRIzol reagent(Invitrogen) according to the protocol of Invitrogen. Preparation ofcDNA by Reverse Transcription Reaction Using Oligo Dt Primers wasperformed with GeneRacer Kit (Invitrogen) according to the protocolprovided by Invitrogen. cDNAs of the variable regions of the H- andL-chains were amplified by PCR using 3′ primer and GeneRacer 5′ primer(5′-CGACTGGAGCACGAGGACACTGA-3′ (SEQ ID NO:14)) attached to GeneRacerKit, each corresponding to mouse constant regions γ1 and κ, with PhusionDNA polymerase (New England Biolabs). The 3′ primer used for PCRamplification of the H-chain variable region (VH) was5′-GCCAGTGGATAGACAGATGG-3′(SEQ ID NO:15). The 3′ primer used for PCRamplification of the L-chain variable region (VL) was5′-GATGGATACAGTTGGTGCAGC-3′(SEQ ID NO:16). The amplified VH and VL cDNAswere subcloned into pCR4Blunt-TOPO vectors (Invitrogen) for sequencing.DNA sequence analyses of the variable regions were carried out withTocore (Menlo Park).

Sequence Analysis Method by Takara (M35A)

A hybridoma cell (M35A) was cultured and amplified. Then, the total RNAof the cell was extracted using RNAiso (Takara Bio Inc.) according toAcid Guanidine-Phenol-Chloroform method (AGPC method). The extracted RNAwas subjected to DNase I treatment according to a common technique, thento phenol chloroform treatment to remove DNase I, and to ethanolprecipitation for purification. The resulting RNA was again suspended indistilled water and used for analysis. About 1 μg of DNase-1-treated RNAas a template and Random Primers (9mer) were used with ReverseTranscriptase M-MLV (RNase H free) for reverse transcription reaction.For PCR amplification of the variable regions, a part of each reversetranscription reactant was used as a template, Heavy Primers 1 and 2(Amersham Bioscience) for H-chain, Light Primer Mix (AmershamBioscience) for L-chain, and TaKaRa LA Taq (Takara) as PCR enzyme wereused.

The DNA fragment obtained by PCR was subjected to electrophoresis withagarose gel. The band was excised out and the gel was eluted to purifythe DNA. The purified DNA was cloned into pMD20-T vector for TA cloning.For DNA sequence analysis of the variable regions, M13-47 primersequence included in pMD20-T vector was used for gene sequencing. Forsequencing reaction, BigDye Terminators v3.1 cycle sequencing kit(Applied Biosystems) was used with ABI3730 sequencer (AppliedBiosystems) according to the protocol of the manufacturer.

As can be appreciated from FIGS. 1A and 1B, although there were slightdifferences in the resulting sequences depending on the analysis method(or the analysis software) employed, there was no difference in theamino acid sequences of CDRs due to different analysis methods.

Example 3 Effect of Anti-Human α9 Integrin Antibody in Inhibiting CellAdhesion

Since α9 integrin binds to a ligand containing extracellular matrix(ECM) such as OPN, fibronectin, tenascin C and VCAM-1 upon celladhesion, inhibition of cell adhesion by the resulting novel anti-humanα9 integrin antibody was examined as inhibition of binding between anα9-integrin-expressing cell (human melanoma cell G361) and the ligand.

SVVYGLR peptide bound to BSA (bovine serum albumin) was used as OPNpeptide, and a protein obtained by expressing the third region ofFibronectin Type III repeat of human tenascin-C in E. coli (where RGDsequence within this region was replaced with RAA sequence) was used asTN-C fn3 (RAA).

OPN peptide or tenascin C fragment (TN-C fn3 (RAA)) (5 μg/mL) was leftin a 96-well plate at 37° C. for an hour, and then blocked with 0.5%BSA/PBS. The human melanoma cells G361 were prepared to be 1×10⁵cells/mL with 0.25% BSA/DMEM medium, and added with the anti-human α9integrin antibody at various concentrations.

Two-hundred μL each of the antibody-added G361 cells was placed into asolid-phased 96-well plate and allowed to react at 37° C. for an hour.Washing was repeated twice with PBS and then the adhered cells wereimmobilized and stained with 0.5% crystal violet/20% methanol. Theresultant was washed with distilled water for three times, dissolved in20% acetic acid and absorbance thereof was determined at 590 nm.Meanwhile, a monoclonal antibody (5A1) against human osteopontin wasused as a negative control while five types of anti-human α9 integrinantibodies prepared beforehand (1K11, 21C5, 24I11, 25B6 and 28S1) wereused as positive controls.

Effect of the anti-human α9 integrin antibody on adhesion of the G361cells to OPN peptide is shown in FIG. 2 and the results with tenascin Cfragments are shown in FIG. 3.

Similar to negative control 5A1 and positive controls 1K11, 25B6 and28S1, M35A had less effect in inhibiting cell adhesion of the G361 cellsto OPN peptide. On the other hand, K33N inhibited cell adhesion with asmaller amount compared to the positive controls 21C5 and 24I11 andshowed an effect of inhibiting cell adhesion to an equal level to thatof Y9A2. As to the adhesion of G361 cells to tenascin C fragment, M35Ahad less effect in inhibiting the cell adhesion while K33N inhibited thecell adhesion with a smaller amount and showed an equal level ofinhibition effect to that of Y9A2, in other words, it showed evidentlystronger inhibition effect than the positive controls 21C5 and 24I11.Hence, K33N, in particular, exerted particularly remarkable effect ininhibiting cell adhesion as compared to other antibodies.

Example 4 Difference in Recognition Sites of Anti-Human α9 IntegrinAntibodies

Since the behavior of newly prepared anti-human α9 integrin antibodyK33N in inhibiting cell adhesion was similar to that of Y9A2,competitive reactions of these antibodies to human α9integrin-expressing cell (hα9/CHO) were detected by FACS to examine thedifference in the recognition sites.

To biotin-labeled K33N or Y9A2 (5 μg/mL, 100 μl), 100 times the amountthereof of K33N, Y9A2 or negative control IgG (0.5 mg/mL, 100 μL) wasadded and then allowed to react with human α9 integrin cells (hα9/CHO,1×10⁷/mL, 100 μL) (4° C., 30 minutes). The cells were washed with FACSbuffer (0.5% BSA/PBS). Streptavidin-labeled APC (0.5 μg/mL, 100 μL) wasadded to the cell solution for reaction (4° C., 20 minutes). Again, thecells were washed with FACS buffer and the dead cells were stained with7-AAD (0.05 mg/mL, 20 μL). Subsequently, the cells were again washedwith FACS buffer and measured by FACS.

As can be appreciated from FIG. 4, when biotin-labeled Y9A2 andnon-labeled Y9A2 were allowed to competitively react with cellsexpressing human α9 integrin, fluorescence-bound cells were obviouslydecreased almost to the background level. In the presence ofbiotin-labeled Y9A2 and non-labeled K33N, however, fluorescence-boundcells were decreased but not as low as the background level. Meanwhile,when biotin-labeled K33N and non-labeled K33N were allowed to competeagainst each other upon addition to the cells expressing human α9integrin, fluorescence-bound cells were obviously decreased butfluorescence-bound cells did not decrease upon addition of non-labeledY9A2 as low as that upon addition of non-labeled K33N. Accordingly,since Y9A2 and K33N did not present complete competition against eachother when they would show competition against each other if theyrecognize the same epitope, Y9A2 and K33N should recognize differentepitopes, suggesting that the antibodies were not identical.

INDUSTRIAL APPLICABILITY

The anti-α9-integrin antibody of the present invention shows anexcellent effect in suppressing a function of α9 integrin and exerts atherapeutic effect against cancers (for example, cancer cellproliferation or metastasis), inflammatory diseases (for example,rheumatoid arthritis, osteoarthritis, hepatitis, bronchial asthma,fibrosis, diabetes, arterial sclerosis, multiple sclerosis, inflammatorybowel diseases (ulcerative colitis, Crohn's disease, etc.)), infectiousdiseases (for example, hepatitis), autoimmune diseases (for example,systemic erythematodes, polymyositis, autoimmune thyroid disease,tubulointerstitial nephritis, myasthenia gravis) and bone diseases (forexample, osteoporosis) and the like. Moreover, a pharmaceuticalcomposition comprising the anti-α9-integrin antibody of the presentinvention as well as the anti-α4-integrin antibody exerts an improvedtherapeutic effect against cancers, inflammatory diseases and the like.The antibody of the present invention may also be utilized as adiagnostic drug since it is capable of pathologically detectingexpression of α9 integrin in cells and tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A A diagram showing the results from analysis of the amino acidsequence of the variable region containing the complementaritydetermining region (CDR) of the heavy chain of anti-human α9 integrinantibodies (K33N and M35A). The results shown were obtained by using twodifferent sequence analysis softwares for K33N and M35A.

FIG. 1B A diagram showing the results from analysis of the amino acidsequence of the variable region containing the complementaritydetermining region (CDR) of the light chain of anti-human α9 integrinantibodies (K33N and M35A). The results shown were obtained by using twodifferent sequence analysis softwares for K33N and M35A.

FIG. 2 A diagram showing the results from examining the effects ofanti-human α9 integrin antibodies (two clones of the present invention(K33N, M35A), additional five clones (1K11, 21C5, 24I11, 25B6 and 28S1)and Y9A2) in inhibiting cell adhesion by using ahuman-α9-integrin-expressing cell (human melanoma cell G361) and anα9-integrin-binding site peptide of OPN (SVVYGLR). A monoclonal antibodyagainst human-osteopontin (5A1) was used as a negative control.

FIG. 3 A diagram showing the results from examining the effects ofanti-human α9 integrin antibodies (two clones of the present invention(K33N, M35A), additional five clones (1K11, 21C5, 24I11, 25B6 and 28S1)and Y9A2) in inhibiting cell adhesion by using ahuman-α9-integrin-expressing cell (human melanoma cell G361) and anα9-integrin-binding site peptide of tenascin C fragment. A monoclonalantibody against human-osteopontin (5A1) was used as a negative control.

FIG. 4 A diagram showing the results from examining the competitivereaction of a novel anti-human α9 integrin antibody (K33N) and Y9A2 to ahuman-α9-integrin-expressing cell.

1-20. (canceled)
 21. An anti-human α9 integrin antibody comprising theamino acid sequences represented by SEQ ID NOS:1, 3, and 5 as CDRH1,CDRH2, and CDRH3, respectively, of the complementary determining regionof the heavy chain (CDRH), and the amino acid sequences represented bySEQ ID NOS: 7, 9 and 11 as CDRL1, CDRL2, and CDRL3, respectively, of thecomplementary determining region of the light chain (CDRL).
 22. Ananti-human α9 integrin antibody comprising amino acid sequencesrepresented by SEQ ID NOS:2, 4, and 6 as CDRH1, CDRH2, and CDRH3,respectively, of the complementary determining region of the heavy chain(CDRH), and the amino acid sequences represented by SEQ ID NOS: 8, 10and 12 as CDRL1, CDRL2, and CDRL3, respectively, of the complementarydetermining region of the light chain (CDRL).
 23. The anti-human α9integrin antibody according to claim 21, wherein the binding between thehuman α9 integrin and the ligand of α9 integrin is inhibited.
 24. Theanti-human α9 integrin antibody according to claim 21, wherein it is amonoclonal antibody.
 25. The anti-human α9 integrin antibody accordingto claim 21, wherein it is a chimeric antibody.
 26. The anti-human α9integrin antibody according to claim 21, wherein it is a humanizedantibody.
 27. The anti-human α9 integrin antibody according to claim 21,wherein it is a human antibody.
 28. An anti-human α9 integrin antibodyproduced by a hybridoma cell assigned Accession No. FERM BP-10830.
 29. Atherapeutic agent for a cancer, an inflammatory disease, an infectiousdisease, an autoimmune disease or a bone disease, comprising theanti-human α9 integrin antibody according to claim 21 as an activecomponent.
 30. A therapeutic agent for a cancer, an inflammatorydisease, an infectious disease, an autoimmune disease or a bone disease,comprising the anti-human α9 integrin antibody according to claim 21 aswell as an anti-human α4 integrin antibody as active components.
 31. Adiagnostic agent for a cancer, an inflammatory disease, an infectiousdisease, an autoimmune disease or a bone disease, comprising theanti-human α9 integrin antibody according to claim 21 as an activecomponent.
 32. A cell that produces the anti-human α9 integrin antibodyaccording to claim
 21. 33. A hybridoma cell assigned Accession No. FERMBP-10830.
 34. An anti-human α9 integrin antibody produced by a hybridomacell assigned Accession No. FERM BP-10831.
 35. A hybridoma cell assignedAccession No. FERM BP-10831.